Role of the Proximal Region of CYP1A2 in the heteromeric P450 complex with POR and Cytochrome‐b5

Abstract

Previous studies have shown that CYP1A2 is capable of forming a CYP1A2‐CYP1A2 homomeric complex that involves the proximal region of the enzyme. Using molecular cloning, specific residues on the proximal face of CYP1A2 (residues L91‐K106: P1 region, residues P131‐S155: P2 region and residues T434‐K447: P3 region) were swapped with homologous residues from CYP2B4 (CYP2B4: T92‐S107 for P1, E132‐E163 for P2 and D438‐R448 for P3). CYP2B4 was chosen as we have evidence that shows that CYP2B4 does not form a homomeric complex in vitro. These constructs were then expressed in HEK‐293T cells and bioluminescence resonance energy transfer (BRET) was used to measure complex formation and disruption. The P1 region of the proximal face was shown to be involved in the homomeric interaction. Interestingly, when we examined the activity of the P1 chimera, lowered activity was observed which suggested that the CYP1A2‐POR (NADPH‐cytochrome P450 reductase) interaction had been affected. POR interacts with P450s on the proximal face. The activity data suggested that the P1 mutation had affected the ability of the chimera to associate with POR. Consequently, BRET competition studies established that the P1 region was involved in the CYP1A2‐POR complex. Subsequent studies with other residues from the proximal face (regions P2 and P3) suggested that these residues were not involved in either the CYP1A2‐CYP1A2 homomeric complex or the CYP1A2‐POR heteromeric complex. We have extended this analysis to examine the effects of these proximal CYP1A2 modifications on its ability to interact with cytochrome b5. BRET competition studies were performed with wild‐type CYP1A2 and wild‑type b5 (tagged with GFP and Rluc respectively) co‐transfected at a high GFP: Rluc ratio with increasing amounts of unlabeled CYP1A2, P1, P2 and P3chimera as the competitor. Preliminary data suggests that the unlabeled P1, P2 and P3chimera were unable to disrupt the wild‐type CYP1A2‐b5 complex as well as the unlabeled CYP1A2, thereby suggesting that the proximal face of CYP1A2 may be involved in the CYP1A2‐ b5 complex. Studies are underway to establish the role of the proximal face in the CYP1A2‐b5 complex and to elucidate the interactions between CYP1A2, POR and cytochrome b5.